Supplemental Materials

This article contains the following supporting material:

• Supplemental Figure 1 - AP-2-GFP is not recruited at sites of FcR-mediated phagocytosis.
  (A) RAW264.7 cells were transiently transfected to express α-adaptin-GFP and after 24h, the cells were incubated for 3 minutes with IgG-SRBCs, fixed and stained with Cy3-anti-rabbit IgG to reveal external SRBCs (middle panel). The cells were then permeabilized and labelled with Alexa350-phalloïdine to detect polymerized actin (right panel). Cells were analyzed by wide-field microscopy. The α-adaptin-GFP appeared as dotted structures, most probably decorating clathrin coated pits at the plasma membrane (left panel). No recruitment of the α adaptin-positive structures was observed under bound particles (arrows). Bar, 5 μm.
  (B) RAW264.7 cells were incubated for 3 min with IgG-SRBCs fixed, permeabilized and stained with anti-γ-adaptin (AP-1) (left pannels) followed by Cy3-anti-mouse Fab'2 and Alexa350-coupled phalloidin to detect phagocytic cups. The arrowhead on the left panel, shows a phagocytic site without AP-1 enrichment. Polymerized actin is shown in the right panel. Bar, 5 μm.
• Supplemental Figure 2 - δ-adaptin is required for efficient FcR-mediated phagocytosis
  (A) RAW264.7 cells were incubated for 3 minutes with IgG-SRBCs, fixed and stained with Cy2-anti-rabbit IgG to reveal external SRBCs (right panel). The cells were then permeabilized and labelled with anti-δ-adaptin followed by Cy3-anti-mouse IgG to detect AP-3 complexes (left panels). Cells were analyzed by wide-field microscopy with deconvolution. Medial optical sections are shown. Arrowheads point to AP-3 recruitment at phagocytic sites. Bar, 5 μm.
  (B) The fluorescence intensities measured for AP-3 and GFP in the phagocytic cups were background substracted and expressed as a percentage of the fluorescence intensities in the cell body as described in materials and methods and in Figure 2. Data obtained for δ-adaptin were compared to and expressed as a percentage of values obtained for GFP in the same regions of interest. Data are the mean ± SEM of 15 independent measurements. AP-3 was not enriched in nascent phagosomes as compared to the cell body, as GFP (p > 0.1).
  (C) RAW264.7 cells were transfected either with δ-adaptin siRNA (AP-3) or with a control siRNA (c). Twenty-four hours after transfection, cell lysates were analyzed by Western blot to detect δ-adaptin (lower panel) and then clathrin heavy chain (upper panel).
  (D) Delta-adaptin depleted cells were incubated for 60 minutes at 37°C with IgG-SRBCs, then fixed and stained with Cy2-anti-rabbit IgG to detect external SRBCs. The efficiencies of association (left) and phagocytosis (right) were calculated as described in Material and Methods in 50 δ-adaptin-depleted (AP-3) or control siRNA-treated cells (control). Results are expressed as a percentage of control cells. The means ± SEM of 3 independent experiments are plotted. Depletion of AP-3 lead to a significant inhibition of phagocytosis (p < 0.05).